Colorado Researchers Build a Phage Library to Target Drug-Resistant Enterococcus, Enterobacter and Klebsiella
For more than 15 years, researchers at the University of Colorado Anschutz School of Medicine have been quietly building something that could become increasingly valuable as antibiotic resistance grows: a library of bacteriophages capable of targeting difficult bacterial pathogens, particularly organisms that normally inhabit the human intestine but can cause severe, sometimes persistent infections when they escape into other parts of the body.
The collection is being developed by the laboratory of Breck Duerkop, an associate professor in the Department of Immunology and Microbiology at CU Anschutz. His group studies the molecular interactions between bacteriophages and bacteria, with a particular focus on antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. The laboratory is also expanding its work toward other problematic Gram-negative pathogens, including Enterobacter hormaechei and Klebsiella oxytoca.
The principle behind the collection is straightforward but clinically important. A bacteriophage that efficiently destroys one bacterial strain may have little or no activity against another strain of the same species. Unlike broad-spectrum antibiotics, phages often recognize highly specific structures on the bacterial surface. A sufficiently diverse library therefore increases the probability that researchers can rapidly identify one or several phages active against a bacterial isolate obtained from a particular infection.
Duerkop’s group currently maintains more than 30 phages capable of infecting enterococci and approximately 15–20 targeting Enterobacter and Klebsiella. Rather than building a collection around large numbers alone, the goal is functional diversity. Some phages can infect multiple strains within a bacterial species, whereas others have extremely narrow host ranges. Maintaining different biological solutions to the same pathogen is particularly useful when bacteria acquire resistance to individual phages.
Many of these viruses begin their journey in untreated wastewater. Sewage contains dense and diverse bacterial communities originating partly from the human intestinal microbiome, making it an unusually productive environment in which to search for phages targeting gut-associated bacteria. Researchers combine wastewater samples with the bacterial strain they want to target and then grow the bacteria as a lawn on agar. When a suitable lytic phage infects and destroys bacterial cells, it produces a clear zone known as a plaque. Individual plaques can then be collected and the corresponding phages propagated and characterized.
After isolation, the CU Anschutz researchers purify the phage preparations, including through centrifugation, before storing them in buffered solutions under refrigeration. According to Duerkop, appropriately stored phages can remain stable for long periods, making it possible to progressively accumulate a collection rather than having to begin a new environmental search each time a resistant infection appears.
That library approach addresses one of the fundamental logistical problems of personalized phage therapy: time. For a critically ill patient with a multidrug-resistant infection, starting from environmental sampling, isolating a new phage and preparing it for therapeutic consideration can be slow. A pre-existing collection creates the possibility of screening a clinical bacterial isolate against dozens of already characterized candidates and identifying matches much earlier.
Enterococci provide a particularly relevant test case. E. faecalis and E. faecium are normal inhabitants of the gastrointestinal tract, but they can become opportunistic pathogens in hospitalized or immunocompromised patients. E. faecium in particular has evolved into a major hospital-associated pathogen with extensive intrinsic and acquired antimicrobial resistance, including widespread vancomycin resistance among clinical lineages.
Duerkop’s research therefore goes beyond simply cataloguing viruses that kill Enterococcus. His laboratory investigates the molecular receptors phages use to infect bacteria, the ways bacteria evolve resistance to them, and whether that evolutionary response can create vulnerabilities to existing antibiotics. This is an important distinction because resistance to phages does not necessarily return the pathogen to exactly its original state.
In earlier work, Duerkop and colleagues demonstrated that lytic phages can select changes in E. faecium that alter susceptibility to antibiotics. Their 2021 study showed that interactions between phage resistance and bacterial cell-envelope biology could increase sensitivity to antimicrobial drugs, including beta-lactams. Such evolutionary trade-offs raise the possibility that a phage might provide two forms of therapeutic pressure: directly killing susceptible bacteria while forcing surviving populations toward phenotypes that are less resistant to conventional drugs.
This is one reason the laboratory emphasizes combinations rather than presenting phages simply as replacements for antibiotics. Phage–antibiotic combinations can potentially attack bacteria through different mechanisms simultaneously, while bacterial adaptation to one treatment may alter susceptibility to the other. These interactions are highly dependent on the phage, bacterial strain, antibiotic and treatment conditions, however, so they have to be demonstrated experimentally rather than assumed.
The CU Anschutz phage collection has already moved beyond laboratory experiments. Phages supplied by Duerkop’s team were used in the treatment of a 57-year-old woman with years of recurrent E. faecium bloodstream infections. Her bacteremia had become particularly difficult to control despite combinations of antibiotics with in vitro activity against the organism. The case was subsequently described scientifically in mBio by researchers from the University of Pittsburgh and the University of Colorado.
The patient's history was unusually severe. She had experienced recurrent E. faecium bloodstream infections since 2013 and eventually developed a period of persistent bacteremia lasting 26 days despite antibiotic treatment. No clear anatomical focus could be identified, and investigators suspected repeated reseeding of the bloodstream from the gastrointestinal reservoir of E. faecium.
After emergency investigational new drug authorization from the U.S. Food and Drug Administration, the researchers added the phage Φ9184 to the existing antimicrobial regimen. The patient received 1 × 10^9 plaque-forming units per dose three times daily through both intravenous and oral routes, an approach intended to target both the bloodstream infection and the suspected intestinal reservoir. Her bacteremia became undetectable within 24 hours after phage administration began.
That striking temporal association should be interpreted carefully. The patient continued receiving antibiotics, so the case cannot establish that the phage alone produced clearance. The infection also later recurred. A second phage, ΦHi3, was subsequently added, and phage treatment continued for months. The published study nevertheless reported fewer hospitalizations and an improvement in quality of life during the treatment period, illustrating both the potential and complexity of personalized phage therapy for recurrent enterococcal infection.
The case also illustrates why maintaining several phages against the same pathogen can matter. Bacteria are capable of evolving phage resistance, just as they evolve antibiotic resistance. A library provides researchers with alternative viruses that recognize different bacterial receptors or use different infection mechanisms, creating options when one phage loses activity against an evolving bacterial population.
This principle is becoming increasingly important as phage therapy moves toward individualized treatment. A clinical laboratory could theoretically isolate the pathogen responsible for an infection and compare it against a panel of phages through plaque assays, liquid infection assays or other susceptibility tests. Active candidates could then undergo additional assessment for host range, genomic safety, potency and compatibility with antibiotics before therapeutic use is considered.
The challenge is that a research phage collection is not automatically a clinical pharmacy. Therapeutic phages require much more than the ability to produce plaques on an agar plate. Candidates intended for patients must be genomically characterized, screened for undesirable genes and adequately purified. Preparations must meet appropriate microbiological and endotoxin specifications, particularly when administered intravenously. Stability, concentration and sterility also have to be controlled, while regulatory authorization depends on the jurisdiction and clinical context.
The Colorado collection should therefore be understood as biological infrastructure: a reservoir of characterized diversity from which potential therapeutic candidates can be selected and studied. Its value grows as researchers learn more about each phage's receptor, host range, resistance pathways and interactions with antibiotics.
Duerkop’s program is also expanding beyond the enterococci that have dominated much of the laboratory's previous research. The CU Anschutz team reports growing interest in Enterobacter hormaechei and Klebsiella oxytoca and has already accumulated roughly 15–20 phages targeting Enterobacter and Klebsiella isolates. Both genera include opportunistic pathogens capable of acquiring extensive antimicrobial resistance, making them logical additions to a library designed around difficult-to-treat infections.
There is a broader biological reason to keep expanding such collections. Environmental phage diversity is enormous, yet only a small fraction of naturally occurring bacteriophages have been isolated, sequenced and experimentally characterized. Even among phages infecting the same bacterial species, different viruses can use distinct receptors and deploy very different molecular mechanisms during infection. Every new isolate therefore potentially adds not merely another entry to a freezer but another biological strategy for overcoming a bacterial cell.
The CU Anschutz project consequently sits at the intersection of fundamental microbiology and personalized medicine. Wastewater provides the raw viral diversity. Bacterial lawns reveal which viruses can kill a target strain. Molecular and genomic studies determine how those viruses function and how bacteria respond. The most promising candidates can then be evaluated with antibiotics and, in exceptional circumstances, considered for patients facing infections for which conventional options have become inadequate.
The strategy also highlights an important reality about the future of personalized phage therapy: the therapeutic infrastructure may need to exist before the patient arrives. When a multidrug-resistant bloodstream infection is progressing, waiting weeks to discover a virus from scratch may be impractical. Diverse, well-characterized phage libraries distributed among research and clinical centers could eventually make matching a pathogen to a candidate phage considerably faster.
For Duerkop, the field remains at an early stage despite more than a century of phage research. Antibiotic-resistant infections continue to create clinical situations in which existing drugs are insufficient, while enormous numbers of potentially useful bacteriophages remain unexplored in environmental reservoirs. His laboratory’s growing collection represents one approach to closing that gap: isolate diversity before it is urgently needed, understand its biology, and preserve it so that the right virus may already be available when a difficult bacterial infection appears.
Sources :
University of Colorado Anschutz School of Medicine — Amber Carlson, A ‘Library’ of Bacteria-Killing Viruses: Harnessing the Power of Phages, September 24, 2026.
https://news.cuanschutz.edu/medicine/bacteria-killing-viruses
Stellfox ME, Fernandes C, Shields RK et al. Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia. mBio. 2024;15:e03396-23.
https://doi.org/10.1128/mbio.03396-23
Canfield GS, Chatterjee A, Espinosa J et al. Lytic Bacteriophages Facilitate Antibiotic Sensitization of Enterococcus faecium. Antimicrobial Agents and Chemotherapy. 2021;65:e00143-21.
https://doi.org/10.1128/AAC.00143-21
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